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| Status |
Public on Dec 21, 2023 |
| Title |
FXS_B_650_RNAseq_Rep2 |
| Sample type |
SRA |
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| Source name |
B-lymphocytes
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| Organism |
Homo sapiens |
| Characteristics |
cell type: B-lymphocytes
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| Growth protocol |
Patient-derived B-lymphocytes were cultured as previously described1. In brief, cells were grown in suspension in RPMI 1640 media (Sigma, R8758) supplemented with 2 mM glutamine, 15 % (v/v) Fetal Bovine Serum, 1 % (v/v) penicillin-streptomycin (Thermo Fisher, 15140122) at 37 °C and 5 % CO2. Cells were passaged every 2-4 days, when they reached a density of approximately 5e5 cells/mL. All cell lines were male.
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| Extracted molecule |
total RNA |
| Extraction protocol |
We isolated total RNA from using the mirVana miRNA Isolation Kit (Thermo Fisher, AM1560) according to the manufacturer’s protocol. We used 100 ng of isolated RNA for RNA-seq library preparation using TruSeq Stranded Total RNA Library Prep Gold (Illumina, 20020598) according to the manufacturer’s instruction. In brief, we removed rRNA from the input RNA, followed by double stranded cDNA preparation using 0.8 U of SuperScript II RT (Thermo Fisher, 4376600) and A-tailing end repair. We ligated cDNA to TruSeq RNA Single Indexes Set A (Illumina, 20020492) to enable multiplex sequencing, followed by one round of of size selection (selecting for 300 bp) and bead clean-up : 42.5 uL of sample was purified with 42 uL of Agencourt AMPure XP beads (Beckman Coulter, A63881), then, 50 uL sample was cleaned with 50 uL Agencourt AMPure XP beads (Beckman Coulter, A63881). We amplified the purified samples by 15 PCR cycles and further purified using Agencourt AMPure XP beads (Beckman Coulter, A63881). We assessed library quality and quantities using the Agilent DNA 1000 reagent kit (Agilent, 5067–1504) on the Agilent Bioanalyzer 2100 (Agilent, 5067–4626) and Qubit high sensitivity RNA assay kit (Thermo Fisher, Q32852), respectively before sequencing on NextSeq500 (Illumina).
We ligated cDNA to TruSeq RNA Single Indexes Set A (Illumina, 20020492) to enable multiplex sequencing, followed by one round of of size selection (selecting for 300 bp) and bead clean-up : 42.5 uL of sample was purified with 42 uL of Agencourt AMPure XP beads (Beckman Coulter, A63881), then, 50 uL sample was cleaned with 50 uL Agencourt AMPure XP beads (Beckman Coulter, A63881). We amplified the purified samples by 15 PCR cycles and further purified using Agencourt AMPure XP beads (Beckman Coulter, A63881). We assessed library quality and quantities using the Agilent DNA 1000 reagent kit (Agilent, 5067–1504) on the Agilent Bioanalyzer 2100 (Agilent, 5067–4626) and Qubit high sensitivity RNA assay kit (Thermo Fisher, Q32852), respectively before sequencing on NextSeq500 (Illumina).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
DESEQ_normalized_counts_WT_Bcell.txt
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| Data processing |
RNA-seq reads were mapped to the hg38 ensembl reference transcriptome for both cDNA and ncRNA using kallisto quant with 100 bootstraps of transcript quantification. Reads were mapped to the ensembl cDNA and ncRNA transcriptomes as described in the kallisto documentation. The resulting quantifications were converted into DESEQ2 format, with transcript level counts mapped to gene level counts in R using the library("tximportData") according to DESEQ2 documentation recommendations. Genes with total counts less than 60 across all samples were dropped from analysis. Differentially called transcripts across the 7 cell lines studied were determined in a pairwise manner using DESEQ2 LRT with adjusted p < 0.005.
Assembly: hg38
Supplementary files format and content: The processed data files include abundance calculations (standard kallisto output, ending in .tsv) which includes the number of counts for each transcript. Additionally, Deseq output processed files includes the number of normalized counts for each transcript
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| Submission date |
Nov 23, 2022 |
| Last update date |
Dec 21, 2023 |
| Contact name |
Jennifer E Phillips-Cremins |
| E-mail(s) |
jcremins@seas.upenn.edu
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| Organization name |
University of Pennsylvania
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| Department |
Bioengineering
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| Street address |
415 Curie Blvd
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| City |
Philadelphia |
| State/province |
Pennsylvania |
| ZIP/Postal code |
19104 |
| Country |
USA |
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| Platform ID |
GPL18573 |
| Series (2) |
| GSE218676 |
Spatially coordinated heterochromatinization of long synaptic genes in fragile X syndrome [RNA-seq] |
| GSE218680 |
Spatially coordinated heterochromatinization of long synaptic genes in fragile X syndrome |
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| Relations |
| BioSample |
SAMN31854660 |
| SRA |
SRX18368319 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6754674_GM04025-rep3_abundance.tsv.gz |
3.1 Mb |
(ftp)(http) |
TSV |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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